Polysaccharide nanoparticles

ABSTRACT

Polysaccharide nanoparticles that are particularly useful in for example drug and agent delivery, tissue-specific targeting, for medical imaging and diagnosis, as well as modifiers of physico-chemical properties. The nanoparticles can be highly-branched glucose homopolymers and can be characterized by a uniform spherical shape. They are monodisperse, hydrophilic and produce low solution viscosities. The nanoparticles are non-toxic, biocompatible and biodegradable. Also, the process of isolation of said polysaccharide nanoparticles from various organisms including, but not limited to, microorganisms such as bacteria and yeasts. Also provided are methods for chemical conjugation of the polysaccharide nanoparticles with various agents. Also provided are examples of use of the polysaccharide nanoparticles and their derivatives as drug delivery systems and fluorescent di-agnostics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 61/016,418, filed Dec. 21, 2007 to Dutcher et al., which is herebyincorporated by reference in its entirety.

BACKGROUND

Nanoparticles are being extensively investigated for their benefits inbiomedical applications such as, for example, therapeutic agents andgene delivery, medical imaging, diagnosis, and tissue targeting.However, for medical applications and especially human health care,there can be stringent material requirements. Some of the more importantrequirements include, for example, low toxicity and biocompatibility ofthe material. Furthermore, it is very desirable for medical applicationsthat nanoparticles be biodegradable, hydrophilic, and non-immunogenic.Monodispersity is another very desirable feature of nanoparticles, sincesize may greatly influence the distribution and accumulation of thenanoparticles in biological tissues, as well as pharmokinetics.Furthermore, nanoparticle surface modification and derivatization occursmuch more predictably if the nanoparticles are monodisperse. However,limited progress has been achieved in identifying suitable nanomaterialswith combination of these and other desirable properties. To date, thereare only a few examples of polymer-based nanoparticles which aremonodisperse and have all of the favorable properties such asnon-toxicity, non-immunogenicity and biodegradability. Common inorganicnanoparticles, such as quantum dots, carbon nanotubes, fullerenes mayhave serious issues with respect to toxicity and biocompatibility.Various polymer based nanoparticles, both synthetic and naturallyextracted, have been researched, but it is technically difficult toproduce polymers in monodisperse form. The most prominent example ofmonodisperse polymeric nanoparticles is that of highly branchedmolecules called dendrimers, with peptide and polysaccharide-baseddendrimers being most suitable for biomedical applications. However, thecost of such dendrimers, especially dendrimers of high molecular weight,presently is prohibitively high due to technical difficulties in theirsynthesis.

SUMMARY

Various embodiments described herein include compositions, individualparticles and nanoparticles and collections of particles andnanoparticles, methods of making and methods of using compositions, andfurther formulations and devices.

For example, one embodiment provides a composition comprisingnanoparticles comprising branched polysaccharide and wherein thenanoparticles are substantially monodisperse in size.

Another embodiment is a composition comprising nanoparticles purifiedfrom a source, wherein the nanoparticles comprise at least one branchedpolysaccharide, and the nanoparticles are substantially spherical andsubstantially monodisperse in size.

Another embodiment is a composition comprising optionally functionalizednanoparticles comprising branched polysaccharide and wherein thenanoparticles are substantially monodisperse in size.

Other embodiments provide for a composition comprising nanoparticlescomprising branched polysaccharide and wherein the nanoparticles aresubstantially monodisperse in size; a method of producing apolysaccharide nanoparticle; a method of derivatizing the polysaccharidenanoparticles; a method of using a composition for drug delivery; amethod of using a composition for diagnosis of a disease or medicalcondition, a method of using the nanoparticle for blood substituteproduct; and a method of using a composition for cosmetic formulation.

Provided herein are methods for producing, isolating and functionalizingmonodisperse polysaccharide nanoparticles (nanoPS) which are non-toxic,biocompatible and biodegradable. Furthermore, the nanoPS can be composedof a high molecular weight glucose homopolymer that is structurallysimilar to glycogen.

NanoPS molecules can be hydrophilic, highly soluble in water and producelow solution viscosities. They can be functionalized and derivatizedusing common carbohydrate chemistry. nanoPS can be produced withpurities that meet the stringent requirements for biomedical polymers,e.g. for enteral and especially for parenteral administration of drugs.Production of nanoPS can be scaled up using fermentation andpurification techniques that have been well developed in thebiotechnological sector which will produce a low cost product that canbe used for applications usually targeted by dendrimer chemistry.

In one aspect, provided herein are monodisperse polysaccharidenanoparticles (nanoPS) that are useful in drug and agent delivery, formedical imaging, molecular diagnostics and molecular targeting, as wellas modifiers of physico-chemical properties. nanoPS molecules comprisingα-D-glucose chains with 1→4 linkage and branching points occurring at1→6 and with a degree of branching having the range of about 6 to about13%, with a structure that is similar to that reported for glycogencontained in animal tissue. nanoPS molecules have a spherical shape asdetermined using dynamic light scattering. The nanoPS molecules are verymonodisperse in molecular weight, with polydispersity index(M_(W)/M_(n)) values that vary between about 1.000 and about 1.100,depending on the source and purification and isolation method. Thecorresponding weight average molecular weight (M_(w)) ranges from about2.00×10⁶ to about 25.00×10⁶ daltons, as determined using size exclusionchromatography (SEC). Depending on the source and purification andisolation method, the nanoPS molecule diameter can be varied from, forexample, about 20 to about 60 nm, or in other embodiments, from about 20nm to about 350 nm, as determined using multi-angle laser lightscattering (MALLS) and atomic force microscopy (AFM). nanoPS is highlysoluble in aqueous solutions and aprotic polar organic solvents. Thecombination in some embodiments of molecule size in the range of tens ofnanometers, high molecular weight, monodispersity and high solubilitycan make nanoPS suitable for a wide range of industrial and biomedicalapplications.

In another aspect, provided herein are methods for producing nanoPSwhich comprise (a) cultivation of microorganisms in appropriate media,followed by (b) isolation of nanoPS according to the proceduresdescribed herein.

In yet another aspect, provided herein are various functional productsprepared by chemical conjugation of nanoPS molecules with various activecompounds and use thereof in various applications, such as drug deliverysystems, MRI/CT contrast agents, fluorescent diagnostics, bloodsubstitute products, and applications in foods and cosmeticformulations.

One or more advantages of at least some of the embodiments describedherein include: (i) particles of nanoPS are non-toxic, biocompatible andbiodegradable and suitable for parenteral administration, e.g., byinjection or by infusion, either transmucosal or inhalational; (ii)nanoPS can be produced at a significantly lower cost compared tosynthetic polysaccharide-based dendrimers; and/or (iii) a broad varietyof microorganisms can be used for the production of nanoPS, such asbacteria, yeasts, microalgae and cyanobacteria. In particular, thenanoparticles can be highly soluble or dispersible and can be engineeredwith well-controlled properties, similar to synthetic polymers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show (A) a size exclusion chromatography (SEC) plot and(B) an atomic force microscopy (AFM) image obtained for nanoPS preparedaccordingly to Example 3. The SEC plot in (A) comprises a single, narrowpeak. The inset in (A) lists parameter values for the nanoPS molecules.The inset in (B) shows the Fast Fourier Transform of the AFM image whichdemonstrates the dense ordered packing of the nanoPS molecules becauseof their high monodispersity.

FIGS. 2A and 2B show (A) a size exclusion chromatography (SEC) plot and(B) an atomic force microscopy (AFM) image obtained for nanoPS preparedaccordingly to Example 2. The SEC plot in (A) comprises a single, narrowpeak. The inset in (A) lists parameter values for the nanoPS molecules.

FIGS. 3A and 3B show (A) a size exclusion chromatography (SEC) plot and(B) an atomic force microscopy (AFM) image obtained for nanoPS preparedusing method accordingly to Example 5. The SEC plot in (A) comprises ofa single, narrow peak. The inset in (A) lists parameter values for thenanoPS molecules.

FIG. 4. GC-MS spectrum of permethylated alditol acetates obtained fornanoPS isolated in Example 3.

FIG. 5. ¹H NMR spectrum obtained at 42° C. for nanoPS isolated inExample 3.

FIG. 6. Dynamic Light Scattering plot of the polysaccharidenanoparticles prepared in accordance with Example 3 of the presentinvention.

FIG. 7. shows a fluorescence microscopy image of polysaccharidenanoparticle-Rhodamine B conjugates from Example 16 (orangefluorescence) taken up by normal murine endothelial cell lines after 16hrs incubation. The polysaccharide nanoparticles were accumulated onlyin the cytoplasm. N: nucleus.

DETAILED DESCRIPTION

Priority U.S. provisional application Ser. No. 61/016,418, filed Dec.21, 2007 to Dutcher et al. is hereby incorporated by reference in itsentirety including the claims, working examples, figures, and othersubsections of the application. Seven other references are cited belowand cited to throughout this application. All references cited hereinare hereby incorporated by reference in their entireties.

Various embodiments described herein relate to polysaccharidenanoparticles (nanoPS) that are useful in drug and agent delivery,tissue-specific targeting, for medical imaging and diagnosis, incosmetic formulations, functional foods, as well as modifiers ofphysico-chemical properties.

Synthesis and characterization of polymers and particles thereof aregenerally known in the art. See for example Billmeyer, Textbook ofPolymer Science, 3^(rd) Ed, Wiley, 1984; Allcock et al., ContemporaryPolymer Chemistry, Prentice-Hall, 1981.

Polymer Material Characterization

Polysaccharides and carbohydrates are widely presented in nature and aregenerally known in the art. See, for example, Bohinski, Modern Conceptsin Biochemistry, 4^(th) Ed., Allyn and Bacon, 1983; Allcock et al.,Contemporary Polymer Chemistry, Prentice-Hall, 1981. Polysaccharides cancomprise single monomer species (homopolymers) or multiple monomerspecies (heteropolymers), and can be linear or branched (see, forexample, Bohinski (1983) and Allcock (1981)). Branched polysaccharidehomopolymers of glucose species are generally known in the art (see, forexample, Alberts et al., Molecular Biology of the Cell, 4^(rd) Ed.,Garland Publishing, 2002). The most prominent examples are glycogen inanimals and amylopectin in plants which both have energy storagefunctions. Both glycogen and amylopectin comprise glucose units whichare linked by α-1,4 glycosidic bonds, and the branching created throughα-1,6 glycosidic bond with a second glucose unit. The degree ofbranching (DB) is given by the ratio of the number of glucose unitswhich have branching points (α-1,6 linkages) to the total number ofglucose units and can be expressed in mol %. It is generally assumedthat amylopectin has lower DB values (3-7 mol %) than glycogen (7-15 mol%), but the values depend on the origin and preparation of the sampleand the experimental method used and therefore differentiation betweenamylopectin and glycogen based on the DB values is elusive. For example,the DB of nanoPS can be within the range of about 6 to about 13 mol %.

The molecular weight of a polymer can be characterized by the weightaverage molecular weight (M_(w)) and the number average molecular weight(M_(n)), and can be measured by methods known in the art including, forexample, light scattering and size exclusion chromatography. Forexample, the M_(w) value of nanoPS can be within the range of about1×10⁶ to about 25×10⁶, or about 2×10⁶ to about 25×10⁶.

The distribution of the molecular weight of polymer molecules ischaracterized by the polydispersity index (PDI) which is defined as theratio of M_(w) to M_(n). For example, nanoPS can have PDI values whichrange from about 1.000 to about 1.300, or about 1.000 to about 1.100.

The polysaccharide nanoparticles can comprise or consist essentially ofother components within the particle beyond the glucose polymer to theextent the basic and novel features described herein are notsubstantially compromised.

Nanoparticle Characterization

Nanoparticles are generally known in the art. See for example Poole etal., Introduction to Nanotechnology, Wiley, 2003; Nanobiotechnology II(Eds. Mirkin and Niemeyer), Wiley-VCH, 2007.

Nanoparticle size, including distributions (dispersity) and averagevalues of the diameter, can be measured by methods known in the art.These primarily include microscopy techniques, e.g. transmissionelectron microscopy and atomic force microscopy. For example, theaverage diameter of nanoPS can be about 20 nm to about 60 nm, or inother embodiments, from about 20 nm to about 350 nm.

It is generally known in the art that nanoparticle systems can becharacterized by low size polydispersity, i.e. monodispersity. See forexample Nanoparticles: From Theory to Application (Ed. Schmid),Wiley-VCH, 2006. The size polydispersity can be described in % by thewidth of the size distribution histogram measured at the 50% of the peakheight divided by mean nanoparticle size and multiplied by 100%. Forexample, the size polydispersity of nanoPS can be from about 4% to about50%.

NanoPS can be used in dispersions and other formulations with use ofsolvent and dispersant systems including aqueous, non-aqueous, and mixedaqueous-nonaqueous systems. Organic solvents can include for examplepolar aprotic solvents, e.g., dimethyl sulfoxide (DMSO), and dimethylformamide (DMF). The pH of the solvent can be for example about3.0-11.0. The concentration of solids in the solution can be for exampleup to 30% (by mass) with no detectable nanoparticles aggregation orprecipitation. nanoPS solutions have no detectable light absorption inthe UV and visible range of wavelengths. Aqueous solutions of nanoPShave low viscosity at relatively high concentrations of up to 30% (bymass).

NanoPS molecules assemble into densely packed, ordered films on variousflat surfaces. The surface of nanoPS molecules can contain severalthousands of terminal hydroxyl functional groups, which can be furthermodified with other functional groups. nanoPS molecules are generallyneutral over a wide range of pH.

Preparation and Isolation of NanoPS

Various embodiments described herein relate to the cultivation ofmicroorganisms under appropriate conditions with a subsequent isolationof nanoPS particles from bacterial biomass. The nanoparticles can bepurified from sources such as biomass including bacterial biomass.

The use of bacteria is preferable since the process can be performed inbatch mode or by using continuous fermentation. This is a scalable andconsistent process, which can be conducted in such a way that it yieldsbiomass which does not have other large molecular weight polysaccharidessuch as amylopectin and amylose, and is free of pathogenic bacteria,parasites, viruses and prions associated with shellfish or animaltissues.

In one embodiment, Gram-negative bacteria are used, which lack thick,rigid cell walls, making the initial step of cell disintegration (beforeextraction procedure) easier or unnecessary.

In one embodiment, rough strains of Gram-negative bacteria are used,which produce no capsular material and which express only roughlipopolysaccharide (LPS), i.e., LPS molecules which lack high molecularweight O-side chains and are terminated only with a coreoligosaccharide. The use of rough strains will decrease the amount ofother high-molecular weight polysaccharides in microbial cells and,therefore, greatly facilitate the separation and purification of nanoPSmolecules.

In one embodiment, rough strains of Escherichia coli, e.g., E. coli K12are used, since these strains have many advantageous characteristics,such as fast growth using inexpensive media, they are accepted for usein the pharmaceutical industry, and the background for large-scalefermentation of these strains is well established. Furthermore, thegenome of this bacterium is completely sequenced and genetic engineeringalterations/manipulations can be performed by those experienced in theart to generate strains which have a high yield of nanoPS.

The amount of nanoPS synthesized by microorganisms depends on thecultivation conditions such as temperature, pH, dissolved oxygenconcentration, growth medium composition, etc. In some instances, theproduction of nanoPS is significantly increased when the growth of themicroorganisms is limited by the absence of certain minerals, such asphosphorus, sulfur, and especially nitrogen, or limited by growthfactors, e.g., essential amino acids.

In one embodiment, E. coli K12 is cultivated using a two stageprocedure. In some instances, the first fermentation is performed in agrowth medium containing all of the necessary mineral elements, and thenthe bacterial cells are transferred into the same growth medium with theexception that the nitrogen source is excluded from the mediumcomposition. Growth in such conditions, with an excess carbon source butlimited by nitrogen, results in a high yield of nanoPS.

One embodiment uses a genetically modified strain of E. coli forcultivation according to the previous embodiment with the aim ofobtaining higher yields of nanoPS.

In another embodiment, a rough strain of Geobacter sulfurreducens isused for nanoPS molecule production. G. sulfurreducens is aGram-negative, strictly anaerobic bacterium which is capable ofanaerobic respiration of fumarate. The medium composition provides anexcess of the carbon source, sodium acetate, which also serves as anelectron donor. However upon bacterial fermentation, an electronacceptor, sodium fumarate, becomes depleted and, therefore, limits thegrowth. This results in a significant increase in nanoPS accumulation inbacterial cells.

After completion of the cultivation process, bacterial cells areseparated from the growth medium by centrifugation or by other meanse.g., by ultrafiltration. This produces wet, concentrated biomass.

Another aspect provides a process for the isolation of nanoPS frombacterial biomass. Although this can be achieved in different ways,variants of the process typically use the following steps:

-   -   1. Cell disintegration by French pressing, or by chemical        treatment, e.g., with phenol;    -   2. Separation of insoluble cell components, e.g., cell walls, by        centrifugation;    -   3. Elimination of proteins and nucleic acids from cell lyzate by        enzymatic treatment followed by dialysis which produces an        extract containing crude polysaccharides and LPS;    -   4. Elimination of LPS by weak acid hydrolysis, or by treatment        with salts of multivalent cations such as Mg²⁺, Al³⁺, etc.,        preferably Ca²⁺, which results in the precipitation of insoluble        LPS products;    -   5. Purification of the nanoPS enriched fraction by dialysis        and/or size exclusion chromatography;    -   6. Precipitation of nanoPS with a suitable organic solvent such        as acetone, methanol, propanol, etc., preferably ethanol.        Alternatively, a concentrated nanoPS solution can be obtained by        ultrafiltration or by ultracentrifugation;    -   7. Freeze drying to produce a powder of nanoPS.

Other types of sources can be used as known in the art. See, forexample, Smith, Biotechnology, 4^(th) Ed., Cambridge University Press,2004. These methods of polysaccharide nanoparticle isolation can beapplied to biological material other than that derived frommicroorganisms. For example, in some embodiments, polysaccharidenanoparticles can be isolated from animals or plants including forexample oysters and rice.

Chemical Functionalization of nanoPS

The present embodiments also provide nanoparticles and molecules withchemically functionalized surface and/or nanoparticles conjugated with awide array of molecules. Chemical functionalization is known in the artof synthesis. See, for example, March, Advanced Organic Chemistry,6^(th) Ed., Wiley, 2007. Functionalization can be carried out on thesurface of the particle, or on both the surface and the interior of theparticle.

Such functionalized surface groups include, but are not limited to,nucleophilic and electrophilic groups, acidic and basic groups,including for example carbonyl groups, amine groups, thiol groups,carboxylic or other acidic groups. Amino groups can be primary,secondary, tertiary, or quaternary amino groups. nanoPS also can befunctionalized with unsaturated groups such as vinyl and allyl groups.

The nanoparticles, as isolated and purified, can be either directlyfunctionalized or indirectly one or more intermediate linkers or spacerscan be used. The nanoparticles can be subjected to one or more than onefunctionalization steps including two or more, three or more, or four ormore functionalization steps.

With functionalization, functionalized nanoPS can be further conjugatedwith various desired molecules, which are of interest for a variety ofapplications, such as biomolecules, small molecules, therapeutic agents,micro- and nanoparticles, pharmaceutically active moieties,macromolecules, diagnostic labels, chelating agents, dispersants, chargemodifying agents, viscosity modifying agents, surfactants, coagulationagents and flocculants, as well as various combinations of thesechemical compounds.

Known methods for polysaccharide functionalization or derivatization canbe used. For example, one approach is the introduction of carbonylgroups, by selective oxidation of glucose hydroxyl groups at positionsof C-2, C-3, C-4 and/or C-6. There is a wide spectrum of oxidativeagents which can be used such as periodate (e.g., potassium periodate),bromine, dimethyl sulfoxide/acetic anhydride (DMSO/Ac₂O) [e.g., U.S.Pat. No. 4,683,298], Dess-Martin periodinane, etc.

nanoPS functionalized with carbonyl groups are readily reactive withcompounds bearing primary or secondary amine groups. This results inimine formation which can be further reduced to amine with a reductiveagent e.g., sodium borohydrate. Thus, the reduction step provides anamino-product that is more stable than the imine intermediate, and alsoconverts unreacted carbonyls in hydroxyl groups. Elimination ofcarbonyls significantly reduces the possibility of non-specificinteractions of derivatized nanoparticles with non-targeted molecules,e.g. plasma proteins.

The reaction between carbonyl- and amino-compounds and the reductionstep can be conducted simultaneously in one vessel (with a suitablereducing agent introduced to the same reaction mixture). This reactionis known as direct reductive amination. Here, any reducing agent, whichselectively reduces imines in the presence of carbonyl groups, e.g.,sodium cyanoborohydrate, can be used.

For the preparation of amino-functionalized nanoPS fromcarbonyl-functionalized nanoPS, any ammonium salt or primary orsecondary amine-containing compound can be used, e.g., ammonium acetate,ammonium chloride, hydrazine, ethylenediamine, or hexanediamine. Thisreaction can be conducted in water or in an aqueous polar organicsolvent e.g., ethyl alcohol, DMSO, or dimethylformamide.

Reductive amination of nanoPS can be also achieved by using thefollowing two step process. The first step is allylation, i.e.,converting hydroxyls into allyl-groups by reaction with allyl halogen inthe presence of a reducing agent, e.g., sodium borohydrate. In thesecond step, the allyl-groups are reacted with a bifunctional aminothiolcompound, e.g., aminoethanethiol [3,4].

Amino-functionalized nanoPS is an important product which are amendableto further modification. For example, amino groups are reactive tocarbonyl compounds (aldehydes and ketones), carboxylic acids and theirderivatives, (e.g., acyl chlorides, esters), succinimidyl esters,isothiocyanates, sulfonyl chlorides, etc.

In certain embodiments, nanoPS molecules are functionalized using theprocess of cyanylation. This process results in the formation of cyanateesters and imidocarbonates on polysaccharide hydroxyls. These groupsreact readily with primary amines under very mild conditions, formingcovalent linkages. Cyanylation agents such as cyanogen bromide, and,preferably, 1-cyano-4-diethylamino-pyridinium (CDAP), can be used forfunctionalization of the nanoPS molecules [5].

Functionalized nanoPS can be directly attached to a chemical compoundbearing a functional group that is capable of binding to carbonyl- oramino-groups. However, for some applications it may be important toattach chemical compounds via a spacer or linker including for example apolymer spacer or a linker. These can be homo- or hetero-bifunctionallinkers bearing functional groups which include, but are not limited to,amino, carbonyl, sulfhydryl, succimidyl, maleimidyl, and isocyanatee.g., diaminohexane, ethylene glycobis(sulfosuccimidylsuccinate)(sulfo-EGS), disulfosuccimidyl tartarate (sulfo-DST),dithiobis(sulfosuccimidylpropionate) (DTSSP), aminoethanethiol, and thelike.

Chemical Compounds and Modifiers for NanoPS/Conjugation

In certain embodiments, chemical compounds which can be used to modifynanoPS include, but are not limited to: biomolecules, small molecules,therapeutic agents, micro- and nanoparticles, pharmaceutically activemoieties, macromolecules, diagnostic labels, chelating agents,dispersants, charge modifying agents, viscosity modifying agents,surfactants, coagulation agents and flocculants, as well as variouscombinations of these chemical compounds.

In certain embodiments, biomolecules used as chemical compounds tomodify nanoPS include, but are not limited to, enzymes, receptors,neurotransmitters, hormones, cytokines, cell response chemical compoundssuch as growth factors and chemotactic factors, antibodies, vaccines,haptens, toxins, interferons, ribozymes, anti-sense agents, and nucleicacids.

In certain embodiments, small molecule chemical compounds used to modifynanoPS result in functionalized nanoPS that is useful for pharmaceuticalapplications and include, but are not limited to, vitamins, anti-AIDSsubstances, anti-cancer substances, antibiotics, immunosuppressants,anti-viral substances, enzyme inhibitors, neurotoxins, opioids,hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants,muscle relaxants and anti-Parkinson substances, anti-spasmodics andmuscle contractants including channel blockers, miotics andanti-cholinergics, anti-glaucoma compounds, anti-parasite and/oranti-protozoal compounds, modulators of cell-extracellular matrixinteractions including cell growth inhibitors and anti-adhesionmolecules, vasodilating agents, inhibitors of DNA, RNA or proteinsynthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal andnon-steroidal anti-inflammatory agents, anti-angiogenic factors,anti-secretory factors, anticoagulants and/or antithrombotic agents,local anesthetics, ophthalmics, prostaglandins, anti-depressants,anti-psychotic substances, anti-emetics and imaging agents.

In certain embodiments, small molecule modifiers of nanoPS can be thosewhich can be useful as catalysts and include, but are not limited to,metal-organic complexes.

In certain embodiments, pharmaceutically useful moieties used asmodifiers for nanoPS include, but are not limited to, hydrophobicitymodifiers, pharmacokinetic modifiers, biologically active modifiers anddetectable modifiers.

In certain embodiments, nanoPS can be modified with chemical compoundswhich have light absorbing, light emitting, fluorescent, luminescent,Raman scattering, fluorescence resonant energy transfer, andelectroluminescence properties.

In certain embodiments, diagnostic labels of nanoPS include, but are notlimited to, diagnostic radiopharmaceutical or radioactive isotopes forgamma scintigraphy and positron emission tomography (PET), contrastagents for Magnetic Resonance Imaging (MRI) (e.g. paramagnetic atoms andsuperparamagnetic nanocrystals), contrast agents for computedtomography, contrast agents for imaging with X-rays, contrast agents forultrasound diagnostic methods, agents for neutron activation, and othermoieties which can reflect, scatter or affect X-rays, ultrasounds,radiowaves and microwaves, fluorophores in various optical procedures,etc. Diagnostic radiopharmaceuticals include gamma-emittingradionuclides, e.g., indium-111, technetium-99m and iodine-131, etc.Contrast agents for MRI (Magnetic Resonance Imaging) include magneticcompounds, e.g. paramagnetic ions, iron, manganese, gadolinium,lanthanides, organic paramagnetic moieties and superparamagnetic,ferromagnetic and antiferromagnetic compounds, e.g., iron oxidecolloids, ferrite colloids, etc. Contrast agents for computed tomographyand other X-ray based imaging methods include compounds absorbingX-rays, e.g., iodine, barium, etc. Contrast agents for ultrasound basedmethods include compounds which can absorb, reflect and scatterultrasound waves, e.g., emulsions, crystals, gas bubbles, etc. Otherexamples include substances useful for neutron activation, such as boronand gadolinium. Further, labels can be employed which can reflect,refract, scatter, or otherwise affect X-rays, ultrasound, radiowaves,microwaves and other rays useful in diagnostic procedures. In certainembodiments a modifier comprises a paramagnetic ion or group.

In certain embodiments, two or more different chemical compounds areused to produce multifunctional derivatives. For example, the firstchemical compound is selected from a list of potential specific bindingbiomolecules, such as antibody and aptamers, and then the secondchemical compound is selected from a list of potential diagnosticlabels.

In certain embodiments, nanoPS molecules can be used as templates forthe preparation of inorganic nanomaterials using methods that aregenerally known in the art (see, for example, Mirkin and Niemeyer, ascited above). This can include functionalization of nanoPS with chargedfunctional groups, followed by mineralization which may includeincubation of functionalized nanoPS in solutions of various cations,e.g. metals, semiconductors. Mineralized nanoPS can be then purified andused in various applications, which include but are not limited tomedical diagnostics, sensors, optics, electronics, etc.

The present description is further expanded with reference to thefollowing non-limiting working examples.

Example 1 Fermentation of G. sulfurreducens PCA

G. sulfurreducens PCA (ATCC 51573) was grown under strict anaerobicconditions at 30° C. for 48 h in modified NBAF prepared according to[7]. The medium contained 15 mM of sodium acetate as electron-donor and40 mM of sodium fumarate as an electron acceptor. Fermentation wascarried out in 15 L vessels, each containing 10 L of the medium. Thefermentation process in each vessel was started with one liter of a 24hour old seed culture. Bacterial cells were harvested by centrifugationat 8,000×g for 15 min and stored at −20° C. The yield was 0.22-0.25 g ofcell dry wt per liter of the growth medium.

Example 2 Isolation of Polysaccharide Nanoparticles from the Biomass ofG. Sulfurreducens PCA by Using Method #1

Method #1 comprises the following steps:a) mixing bacterial biomass with a suitable amount of water to produce asuspension with a final biomass concentration of 10-80 g of dry wt./L,preferably 40 g/L;b) adding 90% (w/v) aqueous phenol to the suspension of bacterial cellsto produce a final phenol concentration of 30-50%, preferably 45%. Thisis followed by vigorous stirring and heating of the suspension to atemperature of 50-68° C., preferably for 10-15 min;c) cooling the mixture to about 0-5° C.;d) centrifuging the suspension (4000-6000×g for 10-20 min at 4° C.),collecting the water fraction, and discarding the insoluble pellet;e) diluting the phenol fraction from step d with pure water by 25-40%(v/v), and repeating steps b, c, and d, pooling the collected waterfractions;f) removing the phenol from the collected water fractions by dialyzingagainst pure water using a membrane with a 12-14 kilodalton molecularweight cut-off for 48-72 h at room temperature;g) adjusting the pH to 8.0 with 0.5M Tris.HCl buffer (pH 8.0), addingmagnesium chloride up to 2 mM, adding DNAse and RNAse, at final enzymeconcentrations of 200 μg/ml and 50 μg/ml respectively, stirring at 37°C. for 1-3 h, centrifuging (50,000×g for 45 min at 4° C.), andcollecting the supernatant;h) centrifuging again (200,000×g for 3 h at 4×C), collecting the pellet;i) adding SDS and Na-EDTA to have final concentrations of 2% and 0.1 M,respectively, adjusting the pH of the mixture to 8.5-9.5 with 0.5M NaOHfollowed by adding proteinase K (50 μg/ml), stirring at 60° C. for 2 h;j) dialyzing against pure water for 24-72 h, and freeze drying;k) dissolving the dried material from the previous step in a 0.5 Msolution of magnesium chloride to the final solid/liquid ratio of1/5-1/6 (wt/vol), centrifuging at 16,000×g for 20 min at 4° C.;l) dialyzing the supernatant for 72 h against water and freeze-drying400 g (wet wt.) of biomass was produced using the procedure in Example 1and it was placed in a 5 L round bottom glass vessel and suspended in1.5 L of nanopure water. Then 1.5 L of 90% (w/v) aqueous phenol wasadded to the suspension. This was followed by vigorous stirring andheating of the suspension to a temperature of 68° C. After 15 min ofstirring, the mixture was cooled to about 0° C. using an ice bath andwas centrifuged at 6000×g at 4° C.

The pellet, containing insoluble cell debris was discarded. Thesupernatant contained two layers: a water fraction and a phenolfraction. The water fraction was collected and kept at 4° C. for furtheruse, while the phenol fraction was re-extracted with ⅓rd volume of purewater under the conditions described above. This operation was repeated3 times before the phenol fraction was discarded. All collected waterfractions were pooled and dialyzed against nanopure water using amembrane with a 12-14 kilodalton molecular weight cut-off for 48-72 h atroom temperature.

Dialysate was supplemented with magnesium chloride (MgCl₂) to make afinal concentration of 2 mM, and the pH was adjusted to 8.0 with a 0.5MTris.HCl buffer. Then the mixture was treated with DNAse and RNAse atfinal enzyme concentrations of 200 μg/ml and 50 μg/ml respectively. Themixture was stirred at 37° C. for 3 h and then centrifuged at 50,000×gfor 45 min at 4° C., collecting the supernatant. The supernatant wascentrifuged again at 200,000×g for 3 h at 4° C., collecting the pellet.The pellet was then resuspended in 2% (w/v) SDS in 0.1M Na₂-EDTA, andthe pH of the mixture was adjusted to 8.5-9.5 using 0.5M NaOH.Proteinase K (25 μg/ml, final concentration) was added to the mixtureand it was stirred at 60° C. for 2 h. Then the mixture was dialyzedagainst nanopure water for 24-72 h at room temperature, changing thewater every 12 h. The dialysate was freeze-dried.

The lyophilized material was dissolved in a 0.5 M solution of magnesiumchloride at a final solid/liquid ratio of 1/6 (wt/vol). The mixture wascooled in the fridge at 4° C. for 24 h and then it was centrifuged at16,000×g for 20 min. The supernatant was dialyzed for 72 h as describedabove and freeze-dried. This method yielded 15 g (dry wt) of nanoPS.

The weight average molar mass moment Mw and polydispersity index (Mw/Mn)of the resultant polysaccharide nanoparticles were 1.270×10̂7 and 1.007as measured using Size Exclusion Chromatography (See FIG. 1A). A Waterschromatography system equipped with a Phenomenex BioSep S4000 column andthree detectors (UV absorption, differential refractive index andmultiple angle laser light scattering (MALLS)) was used.

The diameter and size polydispersity of the resultant polysaccharidenanoparticles were 33.3 nm and 18.2% respectively, as measured usingAtomic Force Microscopy (AFM, see FIG. 1B). To perform AFM measurementsthe nanoparticles were dissolved in ultrapure water (1.0 mg/ml), thenaliquots were dried onto a freshly cleaved mica substrate (approximately1×1 cm). The AFM images were collected using tapping mode.

The mean diameter and size polydispersity of the resultantpolysaccharide nanoparticles were 40.2 nm and 3.5% respectively, asmeasured using a Brookhaven BI-200SM Dynamic Light Scattering systemequipped with a TurboCorr correlator (see FIG. 1C).

Example 3 Isolation of Polysaccharide Nanoparticles from the Biomass ofG. Sulfurreducens PCA Using Method #2

Method #2 for the isolation of polysaccharide nanoparticles from themicrobial biomass comprises the following steps:

a) resuspending the biomass in a solution of 50 mM Tris.HCl (pH 8.0),adding magnesium chloride up to 2 mM, adding DNAse and RNAse (100 μg/mland 25 μg/ml respectively), stirring for 15-30 min at 37° C. to reducethe viscosity;

b) disrupting the microbial cells using a French press (at 15,000lb/in2);

c) adding DNAse and RNAse (to achieve final enzyme concentrations of 200μg/ml and 50 μg/ml respectively), stirring for 15-30 min at 37° C.;

d) centrifuging (21,000×g for 2 h at 4° C.), collecting the supernatant,discarding the pellet containing cell walls, insoluble proteins etc.;

e) adding SDS and Na-EDTA to a final concentration of 2% (w/v) and 0.1 Mrespectively, bringing the pH to 9.0 with 0.1 M NaOH, and treating withproteinase K (up to 200 μg/ml), for 2 h at 60° C.); and dialyzing usinga membrane with a 12-14 kilodalton molecular weight cut-off againstwater to remove proteins and lipids;

f) adding 3 volumes of a solution of magnesium chloride in 95% (v/v)ethanol (0.375 M), stirring and cooling to 0-4° C.; centrifuging(16,000×g for 30 min at 4° C.); keeping the pellet containing nanoPS andLPS;

g) dissolving the pellet in 2% SDS in 0.1 M Na-EDTA (pH 7.5), dialyzingusing a membrane with a 12-14 kilodalton molecular weight cut-offagainst pure water;

h) adding calcium chloride to the solution to a final concentration of0.1-0.2M, and adding ethanol up to 10% (v/v), and centrifuging (16,000×gfor 30 min at 4° C.); keeping the supernanant and discarding the pelletcontaining LPS;

j) adding ethanol or another appropriate solvent to the supernatant toachieve a final solvent concentration in the range of 50-80%; coolingthe mixture to 0-4° C., centrifuging (16,000×g for 20 min at 4° C.);

k) resuspending the pellet containing polysaccharide nanoparticles inwater and dialyzing using a membrane with a 12-14 kilodalton molecularweight cut-off against pure water;

l) freeze drying of the solution containing polysaccharide nanoparticlesto produce a powder.

400 g (wet wt.) of biomass from Example 1 was resuspended in 50 mMTRIS.HCl solution, pH 8.0, supplemented with magnesium chloride to afinal concentration of 2 mM, DNAse and RNAse (100 μg/ml and 25 μg/mlrespectively), and stirred for 15-30 min at room temperature. Thenbacterial cells were disrupted using a French press (at 15,000 lb/in2).

DNAse and RNAse were added to a cell homogenate to achieve final enzymeconcentrations of 200 μg/ml and 50 respectively, followed by stirringfor 2 h at 37 oC. The mixture was centrifuged (16,000×g for 20 min at 4°C.) and the pellet was discarded.

SDS and Na₄-EDTA were added to the supernatant to produce finalconcentrations of 2% (w/v) and 0.1 M respectively, then proteinase K (50μg/ml was added and the solution was stirred for 2 h at 60° C. Themixture was centrifuged (50,000×g for 2 hours at 20° C.) and the pelletwas discarded.

The solution was then dialyzed using a membrane with a 12-14 kilodaltonmolecular weight cut-off against nanopure water for 48 h. The dialyzedsolution was mixed with 3 volumes of pre-cooled 0.375 M solution ofmagnesium chloride in 95% (w/v) ethanol, stirred and cooled to 4° C.using an ice bath. The resulting solution was then centrifuged (16,000×gfor 20 min at 4° C.), the pellet was dissolved in 2% SDS in 0.1 MNa₄-EDTA and dialyzed using a membrane with a 12-14 kilodalton molecularweight cut-off against pure water. Calcium chloride was added to thedialysate to achieve a final concentration of 0.2M CaCl₂ and ethanol upto 10% (v/v), the mixture was left in the fridge for 24 h and then itwas centrifuged (at 16,000×g for 20 min at 4° C.); the supernanant wasretained and the pellet containing LPS was discarded. The supernatantwas mixed with 3 volumes of 95% (w/v) ethanol, cooled to 0° C., andcentrifuged (16,000×g for 20 min at 4° C.). The pellet was resuspendedin water and dialyzed using a membrane with a 12-14 kilodalton molecularweight cut-off against pure water. The dialysate was freeze-dried toproduce a powder of the polysaccharide nanoparticles. The yield was 12.2g of (dry wt.) of polysaccharide nanoparticles.

The weight average molar mass moment M_(w) and polydispersity index(M_(w)/M_(n)) of the resultant nanoparticles were 5.362×10̂6 and 1.031 asmeasured using Size Exclusion Chromatography. A Waters chromatographysystem equipped with a Phenomenex BioSep S4000 column and threedetectors (UV absorption, differential refractive index and multipleangle laser light scattering (MALLS)) was used.

The diameter and size polydispersity of the resultant polysaccharidenanoparticles were 35.3 and 22.7%. respectively, as measured usingAtomic Force Microscopy (AFM, see FIG. 1B). To perform AFM measurementsthe nanoparticles were dissolved in ultrapure water (1.0 mg/ml), thenaliquots were dried onto a freshly cleaved mica substrate (approximately1×1 cm). The AFM images were collected using tapping mode.

The mean diameter and size polydispersity of the resultant nanoparticleswere 60.2 nm and 43.7% respectively as measured using a Wyatt DynaProTitan Dynamic Light Scattering system.

Example 4(a) Fermentation of Escherichia coli K12

E. coli K12 was grown under aerobic conditions at 32° C. for 16 h in asynthetic medium containing 10 g/L of dextrose and 1 g/L of ammoniumsulfate as the sole nitrogen source [6]. Fermentation was carried out ina 15 L fermentor vessel, containing 10 L of the medium with agitation at200 rpm. The fermentation process was started with 100 ml of a 12 hourold seed culture. Bacterial cells were harvested by centrifugation at6,000×g for 15 min and transferred into in a 15 L fermentor vessel,containing 10 L of fresh synthetic medium of the same composition aspreviously described except that the nitrogen source (ammonium sulfate)was excluded. The fermentation continued under the same conditions for 6h and then bacterial cells were harvested by centrifugation at 8,000×gfor 15 min and stored at −20° C.

Example 4(b) Isolation of Polysaccharide Nanoparticles from the Biomassof Escherichia coli K12

E. coli K12 was grown in a synthetic medium containing 20 g/L ofdextrose, 2.5 g/L of ammonium sulfate as the sole nitrogen source, 1.5 gof K₂HPO₄, 0.6 g of KH₂PO₄, 0.2 g magnesium sulfate and 10 mg ofthiamine per liter. One liter of medium was supplemented with 5 mL of atrace element solution containing 1 mol of HCl, 1.5 g of MnCl₂ 4H₂O, 1.0g of ZnSO₄, 0.3 g of H3BO₃, 0.25 g of Na₂MoO₄ 2H₂O, 0.15 g of CuCl₂2H₂O, 0.85 g of Na₂EDTA 2H₂O, 4.0 g of CaCl₂ 2H₂O and 4.5 g of FeSO₄7H₂O per liter. Cultivation was carried out in a 1.5 L fermentationvessel, containing 1.0 L of the medium at 32° C. and constant aeration.The dissolved oxygen concentration was maintained at a minimum of 20% bycontrolling agitation and air flow rate. A sodium hydroxide solution wasused to maintain the pH at 7.2 The fermentation process was started with50 ml of a 12 hour old seed culture. Bacterial cells were harvested atthe early stationary growth phase by centrifugation at 6,000×g for 15min and transferred into a 15 L fermentor vessel, containing 10 L offresh synthetic medium of the same composition as previously describedexcept that the nitrogen source (ammonium sulfate) was excluded. Thefermentation continued under the same conditions for 6 h and thenbacterial cells were harvested by centrifugation at 8,000×g for 15 minand freeze dried. The biomass yield was 2.75 g of dry wt. The biomasswas ground using a mortar and pestle, resuspended in 100 ml of water andthen processed under the conditions described in Example 2. The yield ofpolysaccharide nanoparticles was 0.25 g (dry wt).

The mean diameter and size polydispersity of the resultantpolysaccharide nanoparticles were 40.8 nm and 14.3% respectively, asmeasured using a Wyatt DynaPro Titan Dynamic Light Scattering system.

Example 5 Isolation of nanoPS from Oysters

The oysters were obtained from local grocery store. 100 g of oystertissue (wet wt) was homogenized in a blender and processed as describedin Example 2. The yield of polysaccharide nanoparticles was 1.25 g (drywt).

The weight average molar mass moment M_(w) and polydispersity index(M_(w)/M_(n)) of the resultant polysaccharide nanoparticles extractedfrom oysters were 2.267×10̂7 and 1.099 as measured using Size ExclusionChromatography. A Waters chromatography system equipped with aPhenomenex BioSep 54000 column and three detectors (UV absorption,differential refractive index and multiple angle laser light scattering(MALLS)) was used.

The mean diameter and size polydispersity of the resultantpolysaccharide nanoparticles extracted from oysters were 60.4 nm and30.9% respectively, as measured using a Wyatt DynaPro Titan DynamicLight Scattering system.

Example 6 Characterization of nanoPS from Examples 2, 3, and 5

The nanoPS molecules were dissolved in 0.01M KNO₃ and analyzed using asize exclusion chromatography unit equipped with a Phenomenex BioSepS4000 column and three detectors (UV absorption, differential refractiveindex and multi-angle dynamic laser light scattering (MALLS)). Theresults are shown in FIGS. 1, 2, and 3.

The material was also analyzed using atomic force microscopy (AFM,Dimension 3100 AFM, Veeco Instruments Corp., Santa-Barbara, Calif.)operating in tapping mode using standard silicon cantilevers (AC160TS,force constant 42 N/m, resonance frequency 300 kHz, Al back coating,Olympus, Tokyo, Japan). The nanoPS preparations were dissolved innanopure water (1 mg/ml). Then aliquots were dried onto a freshlycleaved mica substrate (approximately 1×1 cm). Representative AFM imagesare shown on FIGS. 1B, 2B, and 3B. The size of the nanoPS moleculesprepared in examples 2 and 3 was determined to be 33.3 with sizepolydispersity 18.2%, and 35.3 with size polydispersity 22.7%.

Chemical characterization of the structure of the nanoPS molecules wasperformed using gas chromatography mass spectrometry (GC-MS, PolarisQGC-MS FID, Thermo Finnigan, Austin, Tex.) and nuclear magnetic resonancespectroscopy (NMR, Bruker 400 MHz spectrometer). All analysis wasperfomed using D₂O as a solvent.

The sugar composition was analyzed using the alditol-acetate method(GC-MS), and this revealed that nanoPS is a glucose homopolymer.

Permethylated alditol acetate derivatives were used for linkage analysis(GC-MS, electron impact mode). The glucose residues are mainly linkedthrough a 1→4 type linkage and branching occurs predominantly atposition 6. The approximate ratios for the terminal, 1→4 and 1→4,6linked glucose residues are:

-   -   nanoPS isolated in Example 2:1:12.7:1.3 respectively.    -   nanoPS isolated in Example 3:1:11.5:0.8 respectively (See FIG.        4)

Proton NMR revealed one major anomeric peak at 5.41 ppm (a 1→4) and aminor one at 5.02 ppm (α1→4,6). The pattern of the ring region isindicative of a large structure.

NOESY NMR experiments suggested an extremely densely packed molecularstructure.

Example 7 Isolation of Polysaccharide Nanoparticles from the Biomass ofGreenshell Mussels (Perna canaliculus) (New Zealand)

1302 g wet wt. (equal to 257.8 g dry wt.) of Greenshell mussel meat froma local grocery store was mixed with 2.5 L of pure water and homogenizedin a blender at 4° C. for 5 min to an average particle size less than 1mm.

The homogenate was centrifuged at 8000×g at 4° C. and the supernatant(2.5 L) was transferred to a 5 L round bottom glass vessel. Then 0.8 Lof 90% (w/v) aqueous phenol was added to the supernatant. This wasfollowed by vigorous stirring and raising the temperature of thesuspension to 68° C. After stirring at this temperature for 15 min. themixture was cooled to about 4° C. in a refrigerator overnight. Then thewater fraction was collected, while the phenol fraction was discarded.

The water fraction was centrifuged at 8000×g, at 4° C. and the pelletwas discarded. Then ethanol was added to the supernatant to a finalconcentration of 60%, and the mixture was cooled to 4° C. Theprecipitate was isolated by centrifugation (at 6000×g, at 4° C.),resuspended in 0.4 L of water and dialyzed against pure water using a12-14 kDa molecular weight cut-off membrane for 48-72 hrs at roomtemperature, changing the water every 12 hours.

The dialysate was supplemented with magnesium chloride to make a final 2mM MgCl₂ concentration, treated with DNAse and RNAse, at final enzymeconcentrations of 25 μg/ml and 15 μg/ml respectively, at pH 8.0,adjusted with 0.5M Tris*HCl buffer. The mixture was stirred at 37° C.for 3 hrs, then SDS and Na-EDTA were added to have final concentrationsof 2% (w/v) and 0.1 M respectively. The mixture was treated withproteinase K (12 μg/ml) at pH 8.5-9.5, adjusted with 0.5M NaOH, understirring at 60° C. for 2 hours. Then the mixture was dialyzed againstpure water for 24-72 hrs at room temperature, changing the water every12 hours. The dialysate was freeze-dried.

The yield of polysaccharide nanoparticles was 29.7 g (dry wt) whichcorresponds to 11.2% of the mussel meat dry weight.

The weight average molar mass moment M_(w) and polydispersity index(M_(w)/M_(n)) of the resultant polysaccharide nanoparticles extractedfrom Greenshell mussels were 1.444×10̂7 and 1.086 as measured using SizeExclusion Chromatography. A Waters chromatography system equipped with aPhenomenex BioSep S4000 column and three detectors (UV absorption,differential refractive index and multiple angle laser light scattering(MALLS)) was used.

The mean diameter and size polydispersity of the resultantpolysaccharide nanoparticles extracted from Greenshell mussels were 29.7nm and 3.8% respectively, as measured using a Brookhaven BI-200SMDynamic Light Scattering system equipped with a TurboCorr correlator.

Example 8 Conjugation of Polysaccharide Nanoparticles with5-Aminofluorescein Using Cyanylation Chemistry

300 mg of polysaccharide nanoparticles produced according to Example 5was dissolved in 15 ml of pure water and cooled to 4° C. Using 10%sodium carbonate (Na₂CO₃), the pH of the solution was adjusted to 10.8.Then 80 mg of cyanogen bromide (CNBr) in 1 ml of dimethylformamide wasadded, the mixture was stirred and the pH was maintained at 10.8. After4 minutes of stirring, the pH was adjusted to 8.5 with 20% acetic acidand 10 mg of 5-aminofluorescein in 1 ml of dimethylformamide was added.The mixture was stirred at RT for 4 hours in the dark. ThenanoPS-aminofluorescein conjugate was precipitated from the reactionmixture with 3 volumes of cold (0° C.) ethanol. The precipitate wasremoved from the solution by centrifugation at 12000×g at 4° C. for 15min. The pellet was resuspended in 5 ml of water and ethanolprecipitation was repeated another 5 times. Then the product waslyophilized.

Analysis of the conjugate showed the following: from an assay, wemeasured 14 mg of aminofluorescein per 1 g of polysaccharidenanoparticles; the absorbance maximum occurred at 490.5 nm and thefluorescence emission maximum occurred at 517 nm (in a 0.05M potassiumphosphate buffer, pH 7.0).

The mean diameter and size polydispersity of the resultant modifiedpolysaccharide nanoparticles were 48.6 nm and 5.4% respectively, asmeasured using a Brookhaven BI-200SM Dynamic Light Scattering systemequipped with a TurboCorr correlator.

Example 9 Conjugation of Polysaccharide Nanoparticles with DoxorubicinUsing Cyanylation Chemistry 250 mg of polysaccharide nanoparticlesproduced according to Example 5 was dissolved in 10 ml of pure water andcooled to 4° C. Using 10% sodium carbonate (Na₂CO₃), the pH of thesolution was adjusted to 10.8. Then 65 mg of cyanogen bromide (CNBr) in1 ml of DMSO was added, the mixture was stirred and the pH wasmaintained at 10.8. After 4 minutes of stirring, the pH was adjusted to8.5 with 20% acetic acid and 10 mg of doxorubicin hydrochloride in 1 mlof DMSO was added. The mixture was stirred at RT for 4 hours in thedark. The nanoPS-doxorubicin conjugate was precipitated from thereaction mixture with 3 volumes of cold (0° C.) ethanol. The precipitatewas removed from the solution by centrifugation at 12000×g at 4° C. for15 min. The pellet was resuspended in 5 ml of water and ethanolprecipitation was repeated another 5 times. Then the product waslyophilized.

Analysis of the conjugate showed the following: from an assay, wemeasured 15 mg of doxorubicin per 1 g of polysaccharide nanoparticles;the absorbance maximum occurred at 480 nm (in a 0.05M potassiumphosphate buffer, pH 7.0).

The mean diameter and size polydispersity of the resultant modifiedpolysaccharide nanoparticles were 53.3 nm and 55.2% respectively, asmeasured using a Brookhaven BI-200SM Dynamic Light Scattering systemequipped with a TurboCorr correlator.

Example 10 Periodate Oxidation of Polysaccharide Nanoparticles

Polysaccharide nanoparticles (1.0 g), produced according to Example 5,was dissolved in 100 ml of a 0.2M potassium phosphate buffer, pH 7.0,and 0.3 g of sodium periodate in 50 milliliters of water was added tothe solution. The resulting mixture was stirred at room temperature for2 h. Next 5 ml of ethylene glycol was added to quench the reaction. Thenthe solution was dialyzed against nanopure water, using a membrane witha 12-14 kilodalton molecular weight cut-off, for 24 h at roomtemperature. The resulting solution was lyophilized. The yield was of0.81 g, and with the above conditions approximately 5% of the glucoseresidues were oxidized.

The mean diameter and size polydispersity of the resultant modifiedpolysaccharide nanoparticles were 33.8 nm and 30.7% respectively, asmeasured using a Wyatt DynaPro Titan Dynamic Light Scattering system.

However, water solutions of oxidized polysaccharide nanoparticles werenot stable. After two weeks of storage in water at 4° C., more than 85%of oxidized polysaccharide nanoparticles were hydrolyzed as measuredusing Dynamic Light Scattering.

Example 11 Conjugation of Oxidized Polysaccharide Nanoparticles with5-Aminofluorescein

50 mg of oxidized polysaccharide nanoparticles from Example 10 wasdissolved in 4 ml of 0.2 potassium phosphate buffer, pH 7.4. Then 1 mlof 0.5% (w/v) solution of 5-aminofluorescein in 50% (v/v) aqueousethanol was added. The mixture was stirred at room temperature for 48 hin the dark. The polysaccharide nanoparticle—aminofluorescein conjugatewas precipitated from the reaction mixture with 3 volumes of cold (0°C.) ethanol. The precipitate was removed from solution by centrifugationat 12000×g for 15 min at 4° C. The pellet was resuspended in 5 ml ofwater and the ethanol precipitation procedure was repeated 4 times,until all of the unreacted aminofluorescein was washed away, asmonitored by the supernatant absorbance at 487 nm. The washed pellet wasresuspended in 5 ml of 0.2M potassium phosphate buffer, pH 7.4, andsodium borohydride was added to the solution to reach a finalconcentration of 1 mg/ml. The solution was stirred for 15 min and thenanoPS-aminofluorescein conjugate was precipitated as described above.The final product was lyophilized.

Analysis of the conjugate showed the following: Aminofluorescein:glucoseratio of 1:233; absorbance maximum at 490.5 nm; and fluorescenceemission maximum at 517 nm (in a 0.05M potassium phosphate buffer, pH7.0).

The mean diameter and size polydispersity of the resultant modifiedpolysaccharide nanoparticles were 72.8 nm and 31.7% respectively, asmeasured using a Wyatt DynaPro Titan Dynamic Light Scattering system.

However, similarly to oxidized polysaccharide nanoparticles from Example10, water solutions of the 5-aminofluorescein modified polysaccharidenanoparticles were not stable, and after several weeks of storage inwater at 4° C. most of the polysaccharide nanoparticles were hydrolyzedas measured using Dynamic Light Scattering.

Example 12 Conjugation of Oxidized nanoPS with8-Aminonaphthalene-1,3,6-trisulfonic Acid (ANTS)

50 mg of the oxidized nanoPS of Example 10 was dissolved in 4 ml of a0.1 M of sodium borate, pH 8.5 and 1 ml of 1.0% (w/v) ANTS in water andthen 1 ml of 2% (w/v) of sodium cyanoborohyride (NaCNBH₃) in the samebuffer were added to the solution.

The mixture was stirred at 45° C. for 12 h in the dark. The nanoPS-ANTSconjugate was separated from the reaction mixture and washed as wasdescribed in Example 8, with the exception that the ANTS concentrationin the supernatant was monitored by absorbance at 351 nm. The finalproduct was lyophilized.

Analysis of the conjugate showed the following: ANTS:glucose ratio of1:140; absorbance maximum at 354 nm (UV mini 1240 UV-VISspectrophotometer, Shimadzu, Kyoto, Japan); and fluorescence emissionmaximum at 520 nm in a 0.05M potassium phosphate buffer, pH 7.0 (PTIQuantaMaster UV VIS spectrofluorometer, Photon Technology InternationalInc., London, Canada)

Example 13 Conjugation of Oxidized nanoPS with Congo Red

50 mg of oxidized nanoPS of Example 10 was dissolved in 4 ml of a 0.2potassium phosphate buffer, pH 7.4 and 1 ml of 1.0% (w/v) aqueoussolution of Congo Red was added to it. The mixture was stirred at roomtemperature for 48 h in the dark. The nanoPS-Congo Red conjugate wasprecipitated from the reaction mixture with 3 volumes of cold (0° C.)ethanol. The precipitate was removed from solution by centrifugation at12,000×g for 15 min at 4° C. The pellet was resuspended in 5 ml of waterand the ethanol precipitation procedure was repeated 4 times, until allof the unreacted Congo Red was washed away, as monitored by thesupernatant absorbance at 487 nm. The washed pellet was resuspended in 5ml of 0.2M potassium phosphate buffer, pH 7.4, and sodium borohydridewas added to achieve a final concentration of 1 mg/ml. After 15 min ofstirring, the nanoPS-Congo Red conjugate was precipitated as describedabove. The final product was lyophilized. The conjugate yield was 49 mg.

Analysis of the conjugate showed the following: Congo Red:glucose ratioof 1:1300; absorbance maximum at 486.5 nm; and fluorescence emissionmaximum at 580 nm (in a 0.05M potassium phosphate buffer, pH 7.0).

Example 14 Amination of Polysaccharide Nanoparticles

200 mg of polysaccharide nanoparticles, produced according to Example 5,was dissolved in 2 ml of DMSO and 250 mg dry, powdered NaOH was added tothe solution. After 15 min. of stirring, 1.5 ml of 2-bromoethylaminehydro-chloride was added to the reaction mixture (216.66 mg/ml in DMSO).The reaction was allowed to proceed for 4 hrs with constant stirring.After 4 h, 10 ml water was added to the mixture and the aminatedpolysaccharide nanoparticles were precipitated from the solution withethanol (2 volumes of ethanol were added, cooled to 0° C. andcentrifuged at 12000×g for 15 min at 4° C.). The precipitate was placedin water (10 ml) and the ethanol precipitation step was repeated 3 moretimes. The sample was dried and the degree of substitution was estimatedusing proton NMR spectroscopy. According to the NMR data, 5.0 mol % ofthe glucose units were aminated (1 in every 20 sugars).

The mean diameter and size polydispersity of the resultant modifiedpolysaccharide nanoparticles were 25.6 nm and 47.0% respectively, asmeasured using a Wyatt DynaPro Titan Dynamic Light Scattering system.

Example 15 Conjugation of Aminated Polysaccharide Nanoparticles withFluorescamine

17 mg of aminated polysaccharide nanoparticles of Example 14, dissolvedin 8 ml of DMSO was allowed to react with 200 μl of fluorescaminesolution (50 mg/ml in acetone) for 30 min at RT. After 30 min, 16 ml ofwater and 300 μl of 1M CaCl₂ were added to the reaction and thepolysaccharide nanoparticle conjugate was precipitated with 2 volumes ofethanol as described in Example 14. The ethanol precipitation step wasrepeated 3 more times (until the unreacted fluorescamine was washedaway). All of the above steps were performed in the dark. The emissionspectra for the polysaccharide nanoparticle-NH-fluorescamine conjugatewas recorded using a PTI QuantaMaster UV-vis spectrofluorometer (PhotonTechnology International Inc., London, Canada) at an excitationwavelength of 386 nm (100 mM borate buffer, pH 8.5). The degree ofconjugation was calculated as 0.9 mol % (1 in every 111 glucose unitswas conjugated), based on the 380 nm absorbance value (UV mini 1240UV-vis spectrophotometer, Simadzu, Kyoto, Japan).

The mean diameter and size polydispersity of the resultant modifiedpolysaccharide nanoparticles were 34.8 nm and 49.2% respectively, asmeasured using a Wyatt DynaPro Titan Dynamic Light Scattering system.

Example 16 Conjugation of Aminated Polysaccharide Nanoparticles withRhodamine B

25 mg of aminated polysaccharide nanoparticles of Example 14 wasdissolved in 5 ml of a 100 mM carbonate buffer pH 9.6 and 150 μlRhodamine B isothiocyanate solution (100 mg/ml in DMSO) was added. After120 min of stirring at RT, the solution was neutralized with HCl, andthen it was diluted with an additional 5 ml of water and precipitatedwith ethanol as described in Example 14. The ethanol precipitation stepwas repeated 3 more times (until the free dye was washed away). Theprocedure was carried out in the dark. The degree of conjugation is 0.3mol % (calculated from the absorbance value at 540 nm; UV mini 1240UV-vis spectrophotometer, Simadzu, Kyoto, Japan). The rhodamine Bconjugated polysaccharide nanoparticles were used to demonstratepolysaccharide nanoparticle uptake by normal murine endothelial cells(see Example 21).

The size distribution of the resultant modified polysaccharidenanoparticles was bi-modal, with one peak having a mean diameter andsize polydispersity of 30.6 nm and 17.5% respectively, and the otherpeak having a mean diameter and size polydispersity of 124.4 nm and20.0% respectively, as measured using a Wyatt DynaPro Titan DynamicLight Scattering system.

Example 17 Conjugation of Polysaccharide Nanoparticles_withNonenyl-Succinic Anhydride in Pyridine

220 mg of polysaccharide nanoparticles, produced according to Example 5,was made into a suspension in pyridine (three hours mixing at 50° C.)and 1.5 ml n-SA was added to it in 4 portions (during a one hourperiod). The reaction mixture was kept at 50° C., O/N (16 hrs) withstirring. The system was cooled to RT and 4 ml hexane was used toprecipitate the product. The pellet was collected by centrifugation5000×g for 15 min and re-suspended in hexane, then pelleted again usingthe same procedure and this step was repeated two more times. Finallythe pellet was dried and then was placed in 15 ml of water and the pHwas adjusted to 7.0 and lyophilized. The degree of substitution wascalculated from proton NMR spectroscopy data as 3.5 mol % n-SA:nonenyl-succinic anhydride.

The mean diameter and size polydispersity of the resultant modifiedpolysaccharide nanoparticles were 67.4 nm and 28.2% respectively, asmeasured using a Wyatt DynaPro Titan Dynamic Light Scattering system.

Example 18 Conjugation of Polysaccharide Nanoparticles withNonenyl-Succinic Anhydride in Water

2.0 g of polysaccharide nanoparticles, produced according to Example 5,was suspended in 15 ml of water. With a pH electrode inserted into thesolution, the solution was placed into a 32° C. water bath. During a 2 hperiod, 1.0 ml nSA was added to the solution in the following manner:the pH was constantly adjusted to 8.5 with a 4% NaOH solution and nSAwas introduced into the reaction in ˜80 μl portions every 10 min. Afterthe last portion of nSA was added to the solution, the pH keptconstantly monitored and adjusted and the reaction was allowed toproceed for an additional 3 h at which point the pH was changed to 4.0with 1M HCl.

The pellet was centrifuged (12000×g for 15 min). The pellet wasre-suspended in water, the pH was adjusted to 4.0 and the solution wascentrifuged in the same manner two times. Finally, the pellet was takenup in water and dialyzed against water, after the pH was adjusted to7.0.

The mean diameter and size polydispersity of the resultant modifiedpolysaccharide nanoparticles were 34.8 nm and 10.0% respectively, asmeasured using a Wyatt DynaPro Titan Dynamic Light Scattering system.

Example 19(a) Cationic Polysaccharide Nanoparticles:Trimethylaminopropyl-Polysaccharide Nanoparticles

111 mg of polysaccharide nanoparticles, produced according to Example 5,was dissolved in 1.5 ml of DMSO. Then 130 mg NaOH was added and themixture was stirred for one hour. 3 ml of(3-bromopropyl)trimethylammonium bromide solution was added to themixture (64.66 mg/ml in DMSO), the reaction system was kept at 60° C.for 4 h, with constant stirring. After allowing the reaction system tocool to RT, 2 volumes of water (9 ml) and 28 ml of ethanol was added.The mixture was cooled to 4 CC and centrifuged at 12000×g for 15 min at4 C. The pellet was dissolved in water (3 ml), intensively dialyzedagainst water and lyophilized. The degree of substitution was 3.4% asmeasured using NMR spectroscopy.

The mean diameter and size polydispersity of the resultant modifiedpolysaccharide nanoparticles were 49.6 nm and 36.2% respectively, asmeasured using a Wyatt DynaPro Titan Dynamic Light Scattering system.

Example 19(b) Cationic Polysaccharide Nanoparticles:Trimethylamino-Hydroxypropyl-Polysaccharide Nanoparticles

Polysaccharide nanoparticles, produced according to Example 5, weredissolved in DMSO at 74 mg/ml concentration. 50 μl of 4M NaOH was addedto 3.2 ml of the polysaccharide nanoparticle solution and thetemperature was increase to 60° C. 2.1 ml of3-Chloro-2-hydroxypropyltrimethylammonium chloride solution (337 mg/mlconcentration in water) was added in 10 portions (separated by 5minutes) and the reaction was allowed to proceed for 24 h. After coolingthe solution to room temperature, it was neutralized with HCl and theconjugated polysaccharide nanoparticles were precipitated with ethanolas described above. The degree of substitution was 7.1% as measuredusing NMR spectroscopy.

Example 20 Changing the Hydrophilic Character of PolysaccharideNanoparticles by Methylation

Both conjugated and unconjugated polysaccharide nanoparticles weresubjected to permethylation. Using dimethyl sulphoxide as the solvent,solid alkali-metal hydroxide as basic agents and methyl iodide as themethylating agent: 200 mg dry, powdered NaOH was added to 600 ul nanoPSsolution (74 mg/ml in DMSO) and the mixture was stirred at RT for 10min. After 10 min 5 ml CH₃—I solution was given to the reaction mixtureand it was stirred for an additional 2.5 hrs at RT. The solution was“thinned” with 4 ml water and di-chloro-methan (DCM) was introduced tothe system (4:6 volume ratio; DCM: water-DMSO). The O-methylated-nanoPSwas extracted to the DCM phase with thorough mixing and the mixture wascentrifuged on a clinical centrifuge for 10 min, to facilitate phaseseparation. The water layer was removed and replaced with clean d.water. The liquid-liquid extraction was repeated 4 more times. After therepeated extraction process the DCM phase was air dried.

The nanoPS particles appeared fully methylated as the sample wasanalyzed with NMR spectroscopy.

This modification resulted in water-insoluble polysaccharidenanoparticles. The material was nevertheless soluble in dichloromethane.

The effective diameter and size polydispersity of the resultant modifiedpolysaccharide nanoparticles were 340.4 nm and 31.1% respectively, asmeasured in dichloromethane using a Wyatt DynaPro Titan Dynamic LightScattering system. However, we found that methylated polysaccharidenanoparticles produce dynamic complexes with sizes ranging from 100 to500 nm which made measurements in-consistent.

Example 21 Cellular Uptake of Polysaccharide Nanoparticles

Normal murine endothelial cell lines were incubated for 16 hrs withpolysaccharide nanoparticle-Rhodamine B conjugates (1.5 mg/ml),generated using the procedure described in Example 16. Fluorescencemicroscopy demonstrated that polysaccharide nanoparticle-Rhodamine B wastaken up by normal murine endothelial cells. Polysaccharidenanoparticles were accumulated only in cytoplasmic vesicles, with noapparent surface and nucleus staining (See FIG. 7).

Example 22 Toxicological Assessment of Polysaccharide Nanoparticles InVitro

The cellular toxicity of polysaccharide nanoparticles, generatedaccording to Example 5, was compared to that of PLGA(polylactic-co-glycolic acid) nanoparticles that are commonly used indrug delivery systems. In both experiments, Hep2 cells in DMEM medium(100000 cells/ml) were incubated for 24 hrs with differentconcentrations of polysaccharide nanoparticles or PLGA nanoparticles.The number of dead cells as measured using the Trypan blue exclusiontest and the release of LDH (lactate dehydrogenase) showed no noticeabletoxicity of polysaccharide nanoparticles at a concentration of 10 mg/mlthat was 2 orders of magnitude larger than concentrations shown to betoxic for PGLA nanoparticles.

REFERENCES

-   1. Duncan, Nature Reviews Drug Discovery, 2 (2003) pp. 347-360-   2. Manners, Carbohydrate Polymers, 16 (1991) pp. 37-82-   3. Gedda et al. Bioconjugate Chem., 7 (1996) pp. 584-591-   4. Holmberg et al. Bioconjugate Chem. 4 (1993) pp. 570-573-   5. Lees et al. Vaccine 14 (1996) pp. 190-198-   6. Bender, Eur. J. Appl. Microbiol. Biotechnol. 8 (1979) pp. 279-287-   7. Coppi et al., Appl. Environ. Microbiol. 67 (2001) 3180-3187

1. A composition comprising nanoparticles comprising branchedpolysaccharide and wherein the nanoparticles are substantiallymonodisperse in size.
 2. The composition according to claim 1, whereinthe nanoparticles are purified from a source.
 3. The compositionaccording to claim 1, wherein the nanoparticles are furtherfunctionalized.
 4. The composition according to claim 1, wherein thenanoparticles are purified from a source and further functionalized. 5.The composition according to claim 1, wherein the nanoparticles arenon-toxic, biocompatible, and biodegradable.
 6. The compositionaccording to claim 1, wherein the nanoparticles are hydrophilic.
 7. Thecomposition according to claim 1, wherein the polysaccharide structureis substantially similar to that of glycogen, and is substantially freeof amylopectin and amylose.
 8. The composition according to claim 1,wherein the polysaccharide comprises α-D-glucose chains with 1→4 linkageand branching points occurring at 1→6 and with a degree of branchingwithin the range of about 6% to about 13%.
 9. The composition accordingto claim 1, wherein the polysaccharide has a polydispersity index(M_(w)/M_(n)) value between about 1.000 and about 1.100.
 10. Thecomposition according to claim 1, wherein the nanoparticles are purifiedfrom a source; the nanoparticles are non-toxic, biocompatible, andbiodegradable; the nanoparticles are hydrophilic; the nanoparticles aresubstantially similar to glycogen; the homopolymer has a polydispersityindex (M_(w)/M_(n)) value between about 1.000 and about 1.100; thenanoparticles have a spherical shape having a diameter ranging fromabout 20 to about 50 nm (depending on the source, and purification andisolation method); and the nanoparticles have a weight average molecularweight M_(w) ranging from about 2.00×10⁶ to about 25.00×10⁶ Da.
 11. Acomposition comprising nanoparticles purified from a source, wherein thenanoparticles comprise at least one branched polysaccharide, and thenanoparticles are substantially spherical and substantially monodispersein size.
 12. The composition according to claim 11, wherein the sourceis a microorganism source.
 13. The composition according to claim 11,wherein the source is a bacterial source.
 14. The composition accordingto claim 11, wherein the source is a genetically modified bacterialsource.
 15. The composition of claim 11, wherein the polysaccharide hasa polydispersity index (M_(w)/M_(n)) value between about 1.000 and1.100, and wherein the nanoparticles have a diameter ranging from about20 to about 50 nm, and wherein the polysaccharide has a weight averagemolecular weight ranging from about 2.00×10⁶ to about 25.00×10⁶ Da. 16.The composition according to claim 11, wherein the nanoparticles aresubjected to at least one functionalization step.
 17. The compositionaccording to claim 11, wherein the nanoparticles have at least onechemical functionality attached.
 18. The composition according to claim11, wherein the nanoparticles have at least one chemical functionalityattached, and wherein the chemical functionality is carbonyl, amine,hydroxyl, thiol, cyanate ester, imidocarbonate, carboxylic, or otheracidic group, or unsaturated groups such as vinyl and allyl groups. 19.The composition according to claim 11, wherein the nanoparticles have atleast one chemical functionality attached, and wherein the chemicalfunctionality is attached through a homo- or hetero-bifunctional spaceror linker.
 20. The composition according to claim 19, wherein the spaceror linker is diaminohexane, ethylene glycobis(sulfosuccimidylsuccinate)(sulfo-EGS), disulfosuccimidyl tartarate (sulfo-DST),dithiobis(sulfosuccimidylpropionate) (DTS SP), aminoethanethiol.
 21. Acomposition comprising optionally functionalized nanoparticlescomprising branched polysaccharide and wherein the nanoparticles aresubstantially monodisperse in size.
 22. A composition according to claim21, wherein the nanoparticles are purified from a source.
 23. Thecomposition of claim 21, wherein the nanoparticles are functionalized.24. The composition of claim 21, wherein the nanoparticles arefunctionalized with an acidic or basic group.
 25. The composition ofclaim 21, wherein the nanoparticles are functionalized with anelectrophilic or nucleophilic group.
 26. The composition according toclaim 1, wherein the functionalized nanoparticles are modified by smallmolecule, macromolecule, biomolecule, therapeutic agents, microparticle,nanoparticle, pharmaceutically active molecule, diagnostic agent,chelating agent, dispersant, charge modifying agent, viscosity modifyingagent, surfactant, coagulation agent, or flocculant.
 27. The compositionaccording to claim 21, wherein the nanoparticles are functionalized withat least one small molecule, wherein the small molecule is selected fromvitamins, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics and imagingagents.
 28. The composition of claim 21, wherein the nanoparticles arefunctionalized with at least one biomolecule, wherein the biomolecule isselected from enzymes, receptors, neurotransmitters, hormones,cytokines, cell response chemical compounds such as growth factors andchemotactic factors, antibodies, vaccines, haptens, toxins, interferons,ribozymes, anti-sense agents, and nucleic acids.
 29. The composition ofclaim 21, wherein nanoparticles are functionalized with at least onediagnostic label, and the diagnostic label is selected from diagnosticradiopharmaceutical or radioactive isotopes, contrast agents, agents forneutron activation, and other moieties which can reflect, scatter oraffect X-rays, ultrasounds, radiowaves and microwaves, and fluorophores.30. The composition of claim 21, wherein the nanoparticles arefunctionalized with at least one molecule, wherein the molecule haslight absorbing, light-emitting, fluorescent, Raman scattering,fluorescence resonant energy transfer, and electroluminescenceproperties.
 31. A method of producing a polysaccharide nanoparticle,comprising: (a) cultivating a microorganism in a growth medium inabsence of phosphorous, sulfur, and/or nitrogen; and (b) separating thebiomass from the growth medium; (c) isolating the nanoparticle from thebiomass; wherein the nanoparticle is a monodisperse, highly-branchedglucose homopolymer.
 32. The method of claim 31, wherein step (c)comprises: (1) disintegrating the microorganism; (2) obtaining a celllyzate by separating insoluable cell components; (3) obtaining a crudeextract containing polysaccharide nanoparticles by eliminating proteins,nucleic acids, and lipopolysaccharides from the cell lyzate; (4)purifying polysaccharide nanoparticles from the crude extract; and (5)isolating the polysaccharide nanoparticles.
 33. The method of claim 31,wherein the microorganism comprises bacteria, yeasts, microalgae, orcyanobacteria.
 34. The method of claim 31, wherein the microorganismcomprises bacteria.
 35. The method of claim 31, wherein themicroorganism is Gram-negative bacteria or rough strain Gram-negativebacteria.
 36. The method of claim 31, wherein the microorganism isGeobacter sulfurreducens or rough strain Geobacter sulfurreducens. 37.The method of claim 31, wherein the microorganism is a geneticallymodified E. Coli.
 38. The method of claim 31, wherein the microorganismis E. Coli K12.
 39. The method of claim 31, wherein step (b) comprisesusing centrifugation or ultrafiltration.
 40. The method of claim 31,wherein step (c) comprises disintegrating the microorganism which isdone by French pressing or by chemical treatment.
 41. The method ofclaim 32, wherein step (2) comprises centrifugation.
 42. The method ofclaim 32, wherein step (3) comprises enzymatic treatment followed bydialysis.
 43. The method of claim 42, wherein step (3) further comprisesweak acid hydrolysis or treatment with salts of multivalent cationsselected from Mg²⁺, Al³⁺, and Ca³⁺.
 44. The method of claim 32, whereinstep (4) comprises dialysis or size exclusion chromatography.
 45. Themethod of claim 32, wherein step (5) comprises precipitating thepolysaccharide nanoparticle with a suitable organic solvent such asacetone, methanol, ethanol, and propanol.
 46. The method of claim 32,wherein step (5) comprises ultrafiltration or ultracentrifugation. 47.The method of claim 31, wherein the microorganism is bacteria selectedfrom Gram-negative bacteria or rough strain Gram-negative bacteria. 48.The method of claim 45, wherein the bacteria is Geobacter sulfurreducensor rough strain Geobacter sulfurreducens, or E. Coli K12.
 49. A methodof producing a polysaccharide nanoparticle, comprising: (a) cultivatinga microorganism in a growth medium in absence of phosphorous, sulfur,and/or nitrogen; and (b) separating the microorganism cells from thegrowth medium to obtain a biomass; (c) isolating the nanoparticle fromthe biomass; which comprises (1) disintegrate microorganism cells; (2)obtaining a cell lyzate by separating insoluable cell components; (3)obtaining an extract containing crude polysaccharides by eliminatingproteins, nucleic acids, and lipopolysaccharides from the cell lyzate;(4) purifying the crude polysaccharide nanoparticle; and isolating thepolysaccharide nanoparticle. wherein the nanoparticle is a monodisperse,highly-branched glucose homopolymer.
 50. A method of producing apolysaccharide nanoparticle, comprising: (a) cultivating a rough strainbacteria in a growth medium in absence of phosphorous, sulfur, and/ornitrogen; and (b) separating the bacteria cells from the growth mediumto obtain a biomass using centrifugation or ultrafiltration; (c)isolating the nanoparticle from the biomass; which comprises (1)disintegrating the bacteria cells by French pressing or by chemicaltreatment; (2) obtaining a cell lyzate by separating insoluable cellcomponents centrifugation; (3) obtaining an extract containing crudepolysaccharides by eliminating from the cell lyzate, proteins andnucleic acids by enzymatic treatment followed by dialysis, andlipopolysaccharides by weak acid hydrolysis or treatment with salts ofmultivalent cations selected from Mg²⁺, Al³⁺, and Ca³⁺; (4) purifyingthe crude polysaccharide nanoparticle by dialysis or size exclusionchromatography; and (5) isolating the polysaccharide nanoparticle byprecipitating the polysaccharide nanoparticle with a suitable organicsolvent such as acetone, methanol, ethanol, and propanol, orultrafiltration or ultracentrifugation. wherein the nanoparticle is amonodisperse, highly-branched glucose homopolymer.
 51. A polysaccharidenanoparticle made by the method of claim
 31. 52. A polysaccharidenanoparticle made by the method of claim
 49. 53. A polysaccharidenanoparticle made by the method of claim
 50. 54. The polysaccharidenanoparticle of claims 51-53, wherein comprises α-D-glucose chains with1→4 linkage and branching points occurring at 1→6 and with branchingdegree of about 6% to about 13%.
 55. The polysaccharide nanoparticle ofclaim 54, wherein the nanoparticle has a polydispersity index(M_(w)/M_(n)) value between about 1.000 and 1.100.
 56. Thepolysaccharide nanoparticle of claim 54, wherein the nanoparticle has aspherical shape having a diameter ranging from about 20 to about 60 nm.57. The polysaccharide nanoparticle of claim 54, wherein thenanoparticle has a weight average molecular weight ranging from about2.00×10⁶ to about 13.00×10⁶ Da.
 58. A composition comprisingnanoparticles comprising branched polysaccharide and wherein thenanoparticles are substantially monodisperse in size and substantiallyfree of other polysaccharides.
 59. A composition comprisingnanoparticles comprising branched polysaccharide and wherein thenanoparticles are substantially monodisperse in size, and wherein thenanoparticles are substantially free of amylopectin and amylose.
 60. Acomposition comprising nanoparticles comprising branched polysaccharideand wherein the nanoparticles are substantially monodisperse in size,wherein the composition is further substantially free of pathogenicbacteria, parasites, viruses, prions, proteins, nucleic acids, lipidsand lipopolysaccharides.
 61. A composition consisting essentially ofnanoparticles consisting essentially of branched polysaccharide andwherein the nanoparticles are substantially monodisperse in size andsubstantially free of other polysaccharides.
 62. A compositionconsisting essentially of nanoparticles comprising branchedpolysaccharide and wherein the nanoparticles are substantiallymonodisperse in size, and wherein the nanoparticles are substantiallyfree of amylopectin and amylose.
 63. A composition consistingessentially of nanoparticles comprising branched polysaccharide andwherein the nanoparticles are substantially monodisperse in size,wherein the composition is further substantially free of pathogenicbacteria, parasites, viruses, prions, proteins, nucleic acids, lipidsand lipopolysaccharides.
 64. A composition consisting essentially ofnanoparticles consisting essentially of branched polysaccharide, andwherein proteins, nucleic acids, lipids and lipopolysaccharides cannotbe detected by NMR, GC-MS, UV-VIS spectroscopy, size exclusionchromatography, fluorescence spectroscopy, XPS.
 65. A method ofderivatizing the polysaccharide nanoparticles of claims 51-57,comprising chemically functionalizing the nanoparticle so that itssurface bears at least one chemical group such as carbonyl, amine,hydroxyl, thiol, cyanate ester, imidocarbonate, carboxylic or otheracidic group, or unsaturated groups.
 66. The method of claim 65, whereinthe surface functional group is carbonyl, the method comprisingoxidation of glucose hydroxyl groups.
 67. The method of claim 65,wherein the surface functional group is amine, the method comprisingreductive amination.
 68. The method of claim 65, comprising cyanylationof the polysaccharide hydroxyl groups, which forms cyanate ester oriminocarbonate.
 69. The method of claim 65, further comprising modifyingthe nanoparticles with a molecule such as by small molecule,macromolecule, biomolecule, therapeutic agents, microparticle,nanoparticle, pharmaceutically active molecule, diagnostic agent,chelating agent, dispersant, charge modifying agent, viscosity modifyingagent, surfactant, coagulation agent, flocculant, or the combinationthereof.
 70. The method of claim 69, wherein the small molecule isselected from vitamins, anti-AIDS AIDS substances, anti-cancersubstances, antibiotics, immunosuppressants, anti-viral substances,enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines,lubricants, tranquilizers, anti-convulsants, muscle relaxants andanti-Parkinson substances, anti-spasmodics and muscle contractantsincluding channel blockers, miotics and anti-cholinergics, anti-glaucomacompounds, anti-parasite and/or anti-protozoal compounds, odulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics and imagingagents.
 71. The method of claim 69, wherein the biomolecule is selectedfrom enzymes, receptors, neurotransmitters, hormones, cytokines, cellresponse chemical compounds such as growth factors and chemotacticfactors, antibodies, vaccines, haptens, toxins, interferons, ribozymes,anti-sense agents, and nucleic acids.
 72. The method of claim 69,wherein the diagnostic label is selected from diagnosticradiopharmaceutical or radioactive isotopes, contrast agents, agents forneutron activation, and other moieties which can reflect, scatter oraffect X-rays, ultrasounds, radiowaves and microwaves, and fluorophores.73. The method of claim 69, wherein the molecule has light absorbing,light-emitting, fluorescent, Raman scattering, fluorescence resonantenergy transfer, and electroluminescence properties.
 74. Apolysaccharide nanoparticle made by the method of claims 65-69.
 75. Apolysaccharide nanoparticle made by the method of claim
 70. 76. Apolysaccharide nanoparticle made by the method of claim
 71. 77. Apolysaccharide nanoparticle made by the method of claim
 72. 78. Apolysaccharide nanoparticle made by the method of claim
 73. 79. A methodof using a composition for drug delivery, wherein the compositioncomprises optionally functionalized polysaccharide nanoparticles,wherein the nanoparticles are monodisperse, highly-branched glucosehomopolymer.
 80. The method of claim 79, wherein the nanoparticlescomprises α-D-glucose chains with 1→4 linkage and branching pointsoccurring at 1→6 and with branching degree of about 10%, has apolydispersity index (M_(W)/M_(n)) value between about 1.000 and 1.100,a spherical shape having a diameter ranging from about 20 to about 50nm, and a weight average molecular weight ranging from about 2.00×10⁶ toabout 25.00×10⁶ Da.
 81. The method of claim 79, wherein thenanoparticles have functional group selected from carbonyl, amine,hydroxyl, thiol, cyanate ester, imidocarbonate, carboxylic or otheracidic group, and is modified by the small molecule selected fromvitamins, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics and imagingagents.
 82. A method of using the nanoparticle of claims 51-64 for drugdelivery.
 83. A method of using the nanoparticle of claim 74 for drugdelivery.
 84. A method of using a composition for diagnosis of a diseaseor medical condition, wherein the composition comprising optionallyfunctionalized polysaccharide nanoparticles, wherein the nanoparticlesare monodisperse, highly-branched glucose homopolymers.
 85. The methodof claim 84, wherein the nanoparticles comprises α-D-glucose chains with1→4 linkage and branching points occurring at 1→6 and with branchingdegree of about 10%, has a polydispersity index (M_(w)/M_(n)) valuebetween about 1.000 and 1.100, a spherical shape having a diameterranging from about 20 to about 70 nm, and a weight average molecularweight ranging from about 2.00×10⁶ to about 25.00×10⁶ Da.
 86. The methodof claim 84, wherein the nanoparticle has functional group selected fromcarbonyl, amine, hydroxyl, thiol, cyanate ester, imidocarbonate,carboxylic or other acidic group, and is modified by wherein thediagnostic label selected from diagnostic radiopharmaceutical orradioactive isotopes, contrast agents, agents for neutron activation,and other moieties which can reflect, scatter or affect X-rays,ultrasounds, radiowaves and microwaves, and fluorophores.
 87. A methodof using the nanoparticle of claim 74 for diagnosis of a disease ormedical condition.
 88. A method of using the nanoparticle of claim 77for diagnosis of a disease or medical condition.
 89. A method of using acomposition for blood substitute product, wherein the compositioncomprises optionally functionalized polysaccharide nanoparticles,wherein the nanoparticles are monodisperse, highly-branched glucosehomopolymer.
 90. The method of claim 89, wherein the nanoparticlescomprise α-D-glucose chains with 1→4 linkage and branching pointsoccurring at 1→6 and with branching degree of about 10%, has apolydispersity index (M_(w)/M_(n)) value between about 1.000 and 1.100,a spherical shape having a diameter ranging from about 20 to about 70nm, and a weight average molecular weight ranging from about 2.00×10⁶ toabout 25.00×10⁶ Da.
 91. A method of using the nanoparticle of claim 74for blood substitute product.
 92. A method of using a composition forcosmetic formulation, wherein the composition comprises optionallyfunctionalized polysaccharide nanoparticles, wherein the nanoparticlesare monodisperse, highly-branched glucose homopolymers.
 93. The methodof claim 92, wherein the nanoparticles comprise α-D-glucose chains with1→4 linkage and branching points occurring at 1→6 and with branchingdegree of about 10%, has a polydispersity index (M_(w)/M_(n)) valuebetween about 1.000 and 1.100, a spherical shape having a diameterranging from about 20 to about 50 nm, and a weight average molecularweight ranging from about 2.00×10⁶ to about 25.00×10⁶ Da.
 94. A methodof using the nanoparticle of claims 74 for cosmetic formulation.
 95. Thecomposition according to claim 8, wherein the degree of branching rangesfrom about 7% to about 13%.
 96. The composition according to claim 10,wherein the weight-average molecular weight ranges from about 2.00×10⁶to about 13.00×10⁶ Da.
 97. The composition according to claim 15,wherein the weight-average molecular weight ranges from about 2.00×10⁶to about 13.00×10⁶ Da.
 98. The polysaccharide nanoparticle of claim 54,wherein the degree of branching ranges from about 7% to about 13%. 99.The polysaccharide nanoparticle of claim 56, wherein the diameter rangesfrom about 20 to about 50 nm.
 100. The method of claim 80, wherein theweight-average molecular weight ranges from about 2.00×10⁶ to about13.00×10⁶ Da.
 101. The method of claim 85, wherein the weight-averagemolecular weight ranges from about 2.00×10⁶ to about 13.00×10⁶ Da. 102.The method of claim 90, wherein the diameter ranges from about 20 toabout 50 nm.
 103. The method of claim 90, wherein the weight-averagemolecular weight ranges from about 2.00×10⁶ to about 13.00×10⁶ Da. 104.The method of claim 93, wherein the weight-average molecular weightranges from about 2.00×10⁶ to about 13.00×10⁶ Da.